The extracellular 2,3-BDO titer was analyzed by GC-BID (Shimatsu, Japan) using the TG-WAXMS column (Shimatsu). The cells were cultured in 40 ml medium in the shaker flask for 11 days. After harvesting and centrifugation (6,000×g, 15 min), the supernatant was diluted 10-fold and analyzed using GC-BID. Meanwhile, 2,3-BDO standard (Sigma) was diluted to 200, 100, 50, 25, and 10 mg/L and analyzed similarly to establish the standard curve. The 2,3-BDO titers were determined based on the area of the signal and the standard curve.
For the analysis of intracellular metabolites F6P, AcCoA, OAA, and sucrose, the cells were cultured in 40 ml BG-11 medium for 11 days and 1 ml of the supernatant was withdrawn. After centrifugation, the cells were lysed using 100 μL TE buffer containing 2 mg/ml lysozyme in a 37 °C water bath for 20 min, followed by centrifugation (12,000×g, 5 min). Subsequently, 500 μL supernatant was analyzed by HPLC-MS (Shimadzu) using Hypersil™ BDS C18 HPLC Column (5 μm, 10 × 250 mm, Thermo Fisher) and 5% acetonitrile as the mobile phase. The standard F6P, AcCoA, OAA, and sucrose (Sigma) were diluted to 1,000, 500, 100, 10, and 1 mg/L and analyzed similarly to generate the standard curve.
For the analysis of intracellular metabolites F6P, AcCoA, OAA, and sucrose, the cells were cultured in 40 ml BG-11 medium for 11 days and 1 ml of the supernatant was withdrawn. After centrifugation, the cells were lysed using 100 μL TE buffer containing 2 mg/ml lysozyme in a 37 °C water bath for 20 min, followed by centrifugation (12,000×g, 5 min). Subsequently, 500 μL supernatant was analyzed by HPLC-MS (Shimadzu) using Hypersil™ BDS C18 HPLC Column (5 μm, 10 × 250 mm, Thermo Fisher) and 5% acetonitrile as the mobile phase. The standard F6P, AcCoA, OAA, and sucrose (Sigma) were diluted to 1,000, 500, 100, 10, and 1 mg/L and analyzed similarly to generate the standard curve.